Abstract The efficacy of exosomes harvested from mesenchymal stromal cells (MSCs) to enhance recovery has been demonstrated in rodent models of stroke (1-3). Building on this, we showed following cortical injury in monkeys that exosomes harvested from monkey MSCs facilitate recovery within 3-5 weeks(5). Here we propose to now conduct an analysis of the time course and mechanisms underlying exosome-mediated recovery. In previous studies, we harvested brain tissue at 14 weeks post-injury, at which time markers of acute inflammation and repair processes have stabilized. Additionally, our recent findings show a marked difference in the time course of recovery, with exosome-treated monkeys exhibiting full recovery by 3-5 weeks post-injury and untreated animals reaching a plateau in recovery by 8-12 weeks. To better understand the effects on both inflammation and plasticity, we now propose to harvest brains at 4 and 8 weeks post-injury while collecting blood and CSF at multiple time points to investigate temporal changes in biomarkers that underlie recovery. We hypothesize that exosomes will limit acute damage after cortical injury by acting on microglia and other brain cells to promote a switch from pro-inflammatory to anti-inflammatory phenotypes, and transition to a restorative microenvironment. We will test this hypothesis by comparing the recovery of function with and without the administration of exosomes following unilateral cortical injury limited to the hand representation. Following treatment, monkeys will be tested on our motor tasks for either 4 or 8 weeks to assess recovery. After completing testing, monkeys will undergo multi-dimensional diffusion MRI to assess the microstructure of the brain. Brains will then be harvested in order to comprehensively investigate the effect of exosomes on injury-related inflammation and repair processes in several ways. We will assess changes in molecular markers of inflammation, myelin damage, and repair in blood, CSF, and brain tissue and test the effects of exosomes on microglia in vitro. Blood and CSF and brain tissue lysates will be used for ELISA quantification of inflammatory and trophic markers and associated changes in gene expression will be assessed with qPCR. We will also use immunohistochemistry to quantify markers of microglia (LN3, P2YR12, IBA1) and synaptic (VGAT and VGLUTs) and neurite (GAP-43, MAP2) remodeling, and label-free spectral confocal reflectance (SCoRe) microscopy to assess myelin integrity. Then to mechanistically determine the action of exosomes, we will assess the direct effects of exosomes in in vitro, using oxygen glucose deprivation in acute brain slices. In addition, we will assess the effects of exosomes on injury-related changes in neuronal morphology, excitability and signaling by comparing the neurophysiological properties of pyramidal neurons using whole-cell patch-clamp recordings and intracellular filling in acute brain slices. Finally, we will conduct a proteomics analysis of exosomes used in this study to understand the content of exosomes. This project will identify molecules mediating exosome action and generate testable hypotheses for interventions to enhance recovery of function in humans with cortical injury.